Biomedical subjects

T Licht

Publications and source records attributed to T Licht.

34 records · Page 2Linked to original sources

PubMed1996

Characterization of phosphorylation-defective mutants of human P-glycoprotein expressed in mammalian cells.

To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues. Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation. In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with [32P]orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glyco-protein-specific protein kinase purified from multidrug-resistant KB-V1 cells. These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region. Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein.

3T3 Cells↗

PubMed1996

The multidrug-resistance gene in gene therapy of cancer and hematopoietic disorders.

Chemoresistance genes have been identified as an impediment to anticancer drug treatment. In particular, P-glycoprotein, the product of the multidrug-resistance (MDR1) gene, plays a major role in clinical treatment failure. Conversely, expression of an MDR1 cDNA in bone marrow of transgenic animals renders hematopoietic cells chemoresistant. Efficient transfer of drug-resistance genes to normal hematopoietic progenitor cells has been achieved with the use of retroviral vectors. In this article we review approaches which use the multidrug-resistance gene to protect bone marrow from myelosuppression following chemotherapy and as a selectable markerin vivo to increase the expression of nonselectable genes which correct hereditary diseases of the hematopoietic system.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

MDA435/LCC6 and MDA435/LCC6MDR1: ascites models of human breast cancer.

We have established a novel ascites tumour model (MDA435/LCC6) from the oestrogen receptor-negative, invasive and metastatic MDA-MB-435 human breast cancer cell line. MDA435/LCC6 cells grow as both malignant ascites and solid tumours in vivo in nude mice and nude rats, with a tumour incidence of approximately 100%. Untreated mice develop ascites following i.p. inoculation of 1 x 10(6) cells and have a reproducible life span of approximately 30 days, with all animals dying within a 48 h period. The in vivo response of MDA435/LCC6 ascites to several cytotoxic drugs, including doxorubicin, etoposide (VP-16), BCNU and mitomycin C, closely reflects the activity of these single agents in previously untreated breast cancer patients. MDA435/LCC6 cells also retain the anchorage-dependent and anchorage-independent in vitro growth properties of the parental MDA-MB-435 cells, and can be used in standard in vitro drug screening assays. The drug resistance pattern of the MDA435/LCC6 cells suggests that they may have few active endogenous drug resistance mechanisms. To generate a model for the screening of MDR1-reversing agents, MDA435/LCC6 were transduced with a retroviral vector directing the constitutive expression of the MDR1 cDNA, producing a cell line with a classical MDR1 resistance pattern (MDA435/LCC6MDR1). THese ascites models may be a viable alternative to the murine leukaemia ascites (L1210, P388) and, in conjunction with other breast cancer cell lines, facilitate the in vitro and in vivo screening of new cytotoxic drugs and drug combinations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Efficient expression of functional human MDR1 gene in murine bone marrow after retroviral transduction of purified hematopoietic stem cells.

A procedure for efficient transfer of the human MDR1 (multi-drug resistance) gene into murine hematopoietic stem cells was developed. Cells expressing Sca-1 but no lineage-specific or major histocompatibility complex (MHC) class II antigens (Lin-MHC II-Sca-1+) were enriched from 5-fluorouracil-pretreated bone marrow by Ficoll density-gradient and immunomagnetic sorting. Purified cells were cocultured with growth factors and fibroblasts producing replication-deficient retroviruses containing human MDR1 cDNA. Fluorescence-activated cell sorter analysis and rhodamine-123 efflux experiments showed that greater than 60% of cocultured hematopoietic cells expressed functional human P-glycoprotein. After 6 to 8 days, hematopoietic cells were injected intravenously into sublethally irradiated SCID mice. Stem cell properties of the isolated population were confirmed by sustained expression of MDR1 marker cDNA for greater than 4 to 6 months after transplantation, multilineage engraftment, and presence of MDR1 cDNA in bone marrow of secondary recipient mice after retransplantation. Reconstitution of H-2K-mismatched SCID mice showed high engraftment capacity of Lin-MHC II-Sca-1+ cells. MDR1 cDNA was detected in blood of 78% of recipients. P-glycoprotein was expressed in bone marrow of 71% of mice, in both lymphocytes and myelomonocytoid progenitors. P-glycoprotein function in host marrow was confirmed by rhodamine-123 efflux. Transduction of P-glycoprotein may be useful for gene therapy in two ways: to protect bone marrow from myelosuppression after chemotherapy and as a selectable marker in vivo for the introduction of otherwise nonselectable genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Transfer of the MDR1 (multidrug resistance) gene: protection of hematopoietic cells from cytotoxic chemotherapy, and selection of transduced cells in vivo.

Expression of the drug efflux pump P-glycoprotein, encoded by the multidrug resistance (MDR1) gene, has been identified as an impediment to successful chemotherapy of neoplastic diseases. More recently, its potential use for gene therapy has been analyzed. Expression of a full-length MDR1 cDNA in hematopoietic cells renders them resistant to various anticancer drugs, as first shown in a transgenic mouse model. Similarly, mouse hematopoietic progenitor cells in bone marrow or peripheral blood are protected from the toxicity of anticancer chemotherapy by retroviral transduction of the MDR1 gene. Furthermore, cells engineered to express P-glycoprotein survived after the administration of cytotoxic drugs, indicating that the gene could function as a selectable marker in vivo. Recently, MDR1 transduction into isolated pluripotent hematopoietic stem cells has been demonstrated. Clinical studies on MDR1 gene transfer into hematopoietic cells of cancer patients are being planned. Transfer of the MDR1 gene into hematopoietic precursor cells may allow the introduction and selection of otherwise non-selectable genes in bone marrow. The ability to select transduced cells can circumvent the low transduction efficiency that has hampered efficient gene therapy. Recently, fusion genes in which the MDR1 cDNA is fused to genes that correct genetic disorders have been constructed to facilitate gene therapy of inherited metabolic disorders.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Modulation of vindesine and doxorubicin resistance in multidrug-resistant pleural mesothelioma cells by tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to enhance the cytotoxicity of a variety of antineoplastic agents. To examine whether multidrug-resistant cells are targets of TNF-alpha, and whether TNF-alpha is capable of modulating chemoresistance of these cells, a pleural mesothelioma cell line (PXF1118L) and two multidrug-resistant sublines thereof were used as experimental models. Drug resistance of these cells was due to P-glycoprotein expression, as confirmed by (1) staining with a monoclonal antibody (MRK16) specific for human P-glycoprotein, (2) decreased accumulation of [3H]vinblastine that was reversed by verapamil, and (3) enhanced cytotoxicity of vindesine in the presence of verapamil. Parental and multidrug-resistant cells exhibited little but comparable sensitivity to TNF-alpha alone. Combining TNF-alpha with vindesine or, to a lesser extent, with doxorubicin, but not with cisplatin, resulted in greater cytotoxicity towards multidrug-resistant cells than seen for each compound alone, indicating a synergism. In contrast, TNF-alpha failed to modulate vindesine or doxorubicin cytotoxicity in parental cells. [3H]Vinblastine accumulation was unaffected by TNF-alpha, and chemoresistance was reduced by TNF-alpha also in the presence of verapamil (10 microM), indicating that TNF-alpha was acting in a way different from calcium-channel blockers. Though the molecular mechanism by which TNF-alpha was enhancing vindesine and doxorubicin cytotoxicity remained undefined in this study, the numbers of TNF-alpha binding sites on parental and on multidrug-resistant cells were similar, and P-glycoprotein expression was unmodulated during the entire 48 h incubation period. In conclusion, we show that TNF-alpha increases the cytotoxicity of anticancer drugs in multidrug-resistant tumor cells by a mechanism that differs from most chemosensitizing agents, including verapamil. Further studies will be needed to clarify the mechanism by which TNF-alpha synergizes with anticancer drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Ribozyme-mediated cleavage of the MDR-1 transcript restores chemosensitivity in previously resistant cancer cells.

How cancer cells become resistant to chemotherapy is not completely understood, but it is believed that resistance is usually associated with overexpression of drug resistance genes. Drug resistance mediated by the MDR-1 gene is the first well characterized form of drug resistance in human cancer. MDR-1 encodes a phosphoglycoprotein, P-GP, that serves as an energy-dependent drug efflux pump, reducing intracellular drug accumulation and thereby cytotoxicity. We have used ribozymes to reverse the multiple drug resistance phenotype. A hammerhead ribozyme recognizing the GUC sequence at position -6 to -4 close to the translation start site of the 4.5 kb MDR-1 mRNA was prepared by in vitro transcription (MDR-1-RZiv) or chemical synthesis (MDR-1-RZs). Both MDR-1-RZiv and MDR-1-RZs specifically cleaved the MDR-1 mRNA into two parts of the expected size under physiological conditions in an extracellular system with MDR-1-RZiv being more effective. Site-specific cleavage was dependent on time, temperature and [MgCl2]. To examine the in vivo potential of MDR-1-RZ, MDR-1-RZiv and MDR-1-RZs were transfected into a human pleural mesothelioma cell line and into one adriamycin-resistant and one vindesine-resistant subline thereof by liposome-mediated transfer. Incorporation of ribozymes resulted in significantly reduced expression of the MDR-1 gene, with MDR-1-RZs being more potent than MDR-1-RZiv in vitro. MDR-1-RZ reduces P-GP overexpression at the protein level. Liposome-mediated transfer of MDR-1-RZiv or MDR-1-RZs reversed the multiple drug resistance phenotype and restored sensitivity towards chemotherapeutic drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

P-glycoprotein-mediated multidrug resistance in normal and neoplastic hematopoietic cells.

The multidrug transporter, P-glycoprotein (P-gp), is expressed by CD34-positive bone marrow cells, which include hematopoietic stem cells, and in other cells in the bone marrow and peripheral blood, including some lymphoid cells. Multidrug resistance mediated by P-gp appears to be a major impediment to successful treatment of acute myeloid leukemias and multiple myelomas. However, the impact of P-gp expression on prognosis has to be confirmed in several other hematopoietic neoplasms. The role of P-gp in normal and malignant hematopoiesis and clinical attempts to circumvent multidrug resistance in hematopoietic malignancies are reviewed. The recent transduction of the MDR1 gene into murine hematopoietic cells, which protects them from toxic effects of chemotherapy, suggests that MDR1 gene therapy may help prevent myelosuppression following chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Expression of the proliferation-associated Ki-67 antigen of transferrin receptors and of DNA polymerase alpha in human tumour lines: implications for in vitro chemoresistance.

To compare the time course of in vitro expression of various proliferation-associated markers including Ki-67 antigen, transferrin receptors (TfR), and DNA polymerase alpha, six human tumour cell lines of different histological origin were studied under defined conditions. Proliferation markers were demonstrated by peroxidase/anti-peroxidase staining using specific monoclonal antibodies, and their expression was compared to results obtained from [3H]-thymidine incorporation assays and cell counting. Expression of all proliferation markers began to increase during the lag phase, and occurred earlier than elevations of [3H]dT incorporation and cell numbers were recorded. Maximum expression was observed before cell growth reached plateau phase. The time courses of expression of DNA polymerase and Ki-67 were almost identical. The closest correlation of [3H]dT incorporation with time course of expression of proliferation-associated markers was observed, when intranuclear staining of DNA polymerase was analysed. TfR were expressed earlier than the polymerase and Ki-67. Since TfR were also found at remarkable levels in resting cells, they seem less proliferation-specific than Ki-67 and DNA polymerase. While in rapidly growing cell lines more than 95% of the cells expressed Ki-67, TfR, and more than 75% DNA polymerase in cell nuclei, a malignant melanoma and a pleural mesothelioma line displayed fewer than 35% of cells stained for DNA polymerase in cell nuclei during log phase. Determination of growth fractions by monoclonal antibodies may thus contribute to the prediction of chemoresistance by identifying quiescent cells that are not sensitive to S-phase-specific drugs.

Cell Division↗

PubMed1991

Induction of multiple-drug resistance during anti-neoplastic chemotherapy in vitro.

Induction of P-glycoprotein-related multi-drug-resistance (MDR) has been shown in normal and malignant tissues to result from environmental stresses such as heat shock, exposure to carcinogens or X-ray irradiation. To identify conditions under which MDR is enhanced during anti-neoplastic chemotherapy, a cell line showing low-level intrinsic MDR was investigated. In the pleural mesothelioma cell line, PXF1118, less than 1% of cells expressed P-glycoprotein (P-gp), as shown by immunocytochemical staining with monoclonal antibody (MAb) MRK16. Exposure of PXF1118 to vincristine, vindesine, vinblastine or doxorubicin for 2-3 weeks led to an increase in the MDR cell fraction of up to 15-28% during 2 to 3 weeks. For doxorubicin and vindesine, dose-dependence was observed: drug concentrations not capable of eliciting cytotoxicity failed to induce significant P-gp expression. Nutrient starvation in aging medium, exposure to activated cyclophosphamide (even at high concentrations) or cisplatin caused only negligible MDR induction. After exposure to vindesine for 6 weeks, tumor colonies exhibited highly enhanced resistance to Vinca alkaloids, doxorubicin, etoposide and dacarbacine, whereas their sensitivity to mitomycin, activated cyclophosphamide or cisplatin remained unchanged. As determined by [3H]-thymidine uptake and proliferation antigen expression, induction of MDR phenotype was observed at minimal proliferative activity with no change in cell count during exposure to anti-cancer drugs, thus suggesting that the drug treatments changed the phenotype of the cells rather than selecting for a resistant sub-population. In addition, changes in cell differentiation were observed during MDR induction. Induction of P-gp during exposure to anti-cancer drugs thus provides a model for MDR development during initially successful chemotherapy. of P-gp during exposure to anti-cancer drugs thus provides

Adult↗

PubMed1991

Chemotherapy and chemosensitization of transgenic mice which express the human multidrug resistance gene in bone marrow: efficacy, potency, and toxicity.

A common form of multidrug resistance in human cancer results from expression of the MDR1 gene which encodes a plasma membrane energy-dependent multidrug efflux pump. We have engineered transgenic mice which express this multidrug transporter in their bone marrow cells and demonstrated that peripheral WBC of these animals provide a rapid and reliable system for assessing the bioactivity of agents that reverse multidrug resistance. Immunocytochemical analysis of bone marrow smears suggests that the activation of the MDR1 transgene has probably occurred at a very early stage of bone marrow differentiation since most bone marrow cells express the transporter. Expression of this transgene in bone marrow produces about 10-fold resistance to leukopenia induced by taxol compared to normal bone marrow. Chemosensitization of MDR1 mice to daunomycin and taxol, measured by a fall in WBC, is detectable at a dose as low as 0.01 mg/kg R-verapamil. A dose of 0.5 mg/kg R-verapamil reduces the WBC by nearly 50%. Chemosensitization of MDR-transgenic mice with 5 mg/kg R-verapamil, which is highly effective in reversing MDR and readily tolerated by mice, necessitates a reduction of the maximum tolerated dose of most chemotherapeutic agents by only 20%. In addition, detailed histopathological examination shows that treatment of mice with chemotherapeutic drugs and R-verapamil does not change the organ-related toxicity pattern but only moderately accentuates inherent toxic side effects of the chemotherapeutic agents. We conclude that MDR1-transgenic mice represent a valid model for evaluating efficacy, potency, and toxicity associated with chemotherapy and chemosensitization of multidrug-resistant cells in animals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1987

Specificity of monoclonal anti-human insulin antibodies.

To define the specificity and epitope of five monoclonal antibodies (MoAbs) to human insulin, binding studies with artificially modified insulins and a number of native insulins were done. Epitopes on the A-chain (A4, A8-A10) and on the end of the B-chain (B30) could be identified. For two MoAbs, substructures of the amino acid B30 were found, which were essential for binding (hydroxyl and methyl groups of B30). In contrast to most antisera, MoAbs to human insulin show high specificity. However, as the study shows, the specificity is not absolute. With suitable artificial epitope modifications, cross-reaction can be seen. Two of the MoAbs used here show sufficient specificity to discriminate between insulin and proinsulin.

Animals↗

PubMed1986

Hypoglycemia following insulin and proinsulin. A comparison.

The counterregulatory hormonal response to proinsulin-induced hypoglycemia was investigated in eight volunteers. Proinsulin cleared slower from the circulation than insulin. Hypoglycemia occurred slower (2P less than 0.005) and was prolonged, while the overall hypoglycemic activities were comparable. The antilipolytic effect of proinsulin was also prolonged (2P less than 0.001). The response of epinephrine to hypoglycemia was less pronounced after proinsulin (2P less than 0.05). The amount of epinephrine was correlated to the rate of fall in plasma glucose (P less than 0.005). The production of lactate induced by beta-stimulation was also correlated to the fall of glucose (P less than 0.005). The responses of prolactin (2P less than 0.02), norepinephrine (2P less than 0.02), cortisol, and growth hormone were attenuated following proinsulin. The decreases of serum potassium and serum phosphate (2P less than 0.05) were less pronounced. Symptoms like sweating (2P less than 0.01) and dizziness (2P less than 0.01) were milder after proinsulin. It is concluded that the rate of fall in glucose concentration determines the differing counterregulatory responses. We don't relate the differing counterregulatory responses to special insulin-like properties of proinsulin, but to the slower kinetics which is emphasized by the intravenous bolus injection.

3-Hydroxybutyric Acid↗

PubMed1985

Insulin and anti-insulin antibody interaction. Evidence for the formation of 7 S and 10 S structures.

The clearing of monoclonal and polyclonal and anti-insulin antibodies from homogeneous solutions at 100,000 X g was used to estimate the size of soluble insulin-antibody complexes at physiologic concentrations. Monoclonal antibodies cleared as a uniform population of 6.6 S independent of the insulin concentration. Polyclonal antibodies cleared as 6.6 S monomers at saturation and as 10 S particles when the amount of insulin bound decreased, suggesting that a soluble complex with two antibodies was formed. An increase of the affinity and a decrease of antibody valency can be related to the complex formation. The binding affinity of polyclonal sera depends on the composition of the affinities of the IgG monomers and on their ability to form 10 S complexes. The formation of insulin-antibody dimers precludes cross-linking and precipitation. Both types of insulin-antibody complexes have been found in the sera from patients treated with bovine insulin.

Animals↗

PubMed1985

Recognition of human insulin and proinsulin by monoclonal antibodies.

High-affinity monoclonal antibodies (MAB) were obtained from lymph node cell fusions. Affinities ranging from 0.8 X 10(9) L/M to 5.2 X 10(9) L/M were calculated from binding studies with monoiodinated human, bovine, and porcine insulins and human proinsulin. Two monoclonal antibodies were specific for human insulin, recognizing an epitope involving the amino acid B-30 (Thr). Another two monoclonal antibodies were bound to the C-terminal end of the B-chain near B-30. The B-chain-specific monoclonal antibodies did not bind human proinsulin. One monoclonal antibody recognized the A-chain loop in the positions A-8 to A-10. This antibody bound also to human proinsulin. It was concluded that the A-chain loop is exposed on the surface of proinsulin, while the C-terminal B-chain is not available for binding. The study shows that monoclonal antibodies can be used to characterize structures of insulin and proinsulin. In contrast to x-ray studies, the molecules can be used at low concentrations in soluble form. It is suggested to use monoclonal antibodies for the screening of atypical insulins in the serum of diabetic patients and for the further refinement of insulin and proinsulin measurements.

Animals↗

PubMedDate not supplied

Influence of beta-adrenergic antagonists, H1-receptor blockers, analgesics, diuretics, and quinolone antibiotics on the cellular accumulation of the anticancer drug, daunorubicin: P-glycoprotein modulation.

BACKGROUND: Treatment of patients with several drugs simultaneously may result in modulation of the naturally expressed P-glycoprotein (Pgp) at different tissues. With this possibility in mind, we have assessed the ability of different classes of drugs to modulate Pgp function in vitro. Modulation of the Pgp function was studied at in vitro drug concentrations comparable to therapeutic blood levels of the drugs. MATERIALS AND METHODS: Human blood brain barrier endothelial cells and human colon adenocarcinoma cells were transduced or transfected with the multidrug resistance gene (MDR1) to express Pgp. The uptake of fluorescent substrates of Pgp, Rhodamine 123 and daunorubicin, into these cells and NIH3T3/MDR1 and MDCK/MDR1 cells was measured by flow cytometry and in monolayers in the presence and absence of the different drugs. RESULTS: From the tested six H1-receptor blockers, seven beta-adrenergic antagonists, four analgesics, ten diuretics and five quinolons, five drugs inhibited Pgp at therapeutic blood levels and two at somewhat higher concentrations. Significant synergism for blocking Pgp could be demonstrated for several drugs. CONCLUSION: We conclude that administration of several drugs which modulate the function of Pgp to patients may adversely affect the natural function of this efflux pump and may cause drug-drug interactions induced side effects.

3T3 Cells↗